Chapter 19
PROTOCOL
PNA-FISH
WILLIAM M. STRAUSS
Introduction
Peptide nucleic acids (PNAs) are nucleic acid mimics that contain a pseudo-peptide backbone composed of charge neutral and achiral N-(2-aminoethyl) glycine units to which the nucleotides are attached via a methylene carbonyl linker (Nielsen et al. 1991; Egholm et al. 1992; 1993). PNAs
hybridize with high affinity to complementary DNA sequences, forming
PNA-DNA complexes via Watson-Crick or Hoogsteen binding (Leijon et
al. 1994). The PNAIDNA duplex has a higher melting temperature, Tm,
than the DNAIDNA duplex. For instance, a typical IS-mer PNA/DNA
melts at 69°C whereas the corresponding DNAIDNA duplex melts at
S4°C (Egholm et al. 1993). In addition to the high thermal stability of complexes, PNA-DNA binding is highly sensitive to mismatches (Nielsen et al.
1993; Egholm et al. 1993; Jensen et al. 1997). The Tm changes caused by
single base mismatch averaged IS and 11°C, respectively, in PNA/DNA
and DNA/DNA duplexes for a IS-mer oligo. These novel characteristics
of short PNA oligomers obviously lead to development of PNA fluorescence in situ hybridization (PNA-FISH).
Lansdrop et al. (1996) was the first to use directly fluorescein-labeled
(C3TA2h PNA 18-mer for fluorescence in situ hybridization on telomeres.
Since then, PNA-FISH has been applied in detecting trinucleotide repeats
(Taneja 1998) and centromeric repeats (Chen et al. 1999,2000). PNA-FISH
can discriminate between two CENP-B sequences that differ by a single
base pair in mouse and human centromeres. Interestingly, PNA probes
have been used in a flow cytometry/FISH method for determining the replication history and potential replication capacity of several different
types of cells from the same tissue (Hultdin et al. 1998; Rufer et al. 1998).
William M. Strauss, Harvard Medical School, Beth Israel Deaconess Medical Center,
Harvard Institute of Human Genetics, 4 Blackfan Circle, Boston, MA, 02115, USA
(phone +617-432-8475; fax +617432-8476; e-mail wstrauss@hihg.med.harvard.edu)
PROTOCOL
PNA-FISH
WILLIAM M. STRAUSS
Introduction
Peptide nucleic acids (PNAs) are nucleic acid mimics that contain a pseudo-peptide backbone composed of charge neutral and achiral N-(2-aminoethyl) glycine units to which the nucleotides are attached via a methylene carbonyl linker (Nielsen et al. 1991; Egholm et al. 1992; 1993). PNAs
hybridize with high affinity to complementary DNA sequences, forming
PNA-DNA complexes via Watson-Crick or Hoogsteen binding (Leijon et
al. 1994). The PNAIDNA duplex has a higher melting temperature, Tm,
than the DNAIDNA duplex. For instance, a typical IS-mer PNA/DNA
melts at 69°C whereas the corresponding DNAIDNA duplex melts at
S4°C (Egholm et al. 1993). In addition to the high thermal stability of complexes, PNA-DNA binding is highly sensitive to mismatches (Nielsen et al.
1993; Egholm et al. 1993; Jensen et al. 1997). The Tm changes caused by
single base mismatch averaged IS and 11°C, respectively, in PNA/DNA
and DNA/DNA duplexes for a IS-mer oligo. These novel characteristics
of short PNA oligomers obviously lead to development of PNA fluorescence in situ hybridization (PNA-FISH).
Lansdrop et al. (1996) was the first to use directly fluorescein-labeled
(C3TA2h PNA 18-mer for fluorescence in situ hybridization on telomeres.
Since then, PNA-FISH has been applied in detecting trinucleotide repeats
(Taneja 1998) and centromeric repeats (Chen et al. 1999,2000). PNA-FISH
can discriminate between two CENP-B sequences that differ by a single
base pair in mouse and human centromeres. Interestingly, PNA probes
have been used in a flow cytometry/FISH method for determining the replication history and potential replication capacity of several different
types of cells from the same tissue (Hultdin et al. 1998; Rufer et al. 1998).
William M. Strauss, Harvard Medical School, Beth Israel Deaconess Medical Center,
Harvard Institute of Human Genetics, 4 Blackfan Circle, Boston, MA, 02115, USA
(phone +617-432-8475; fax +617432-8476; e-mail wstrauss@hihg.med.harvard.edu)